The hematopoietic stem cell (HSC) is a pluripotent progenitor cell that has been characterized as a cell that is transplantable and can self-replicate or generate daughter cells that are destined to commit to mature cells of different specific lineages.
Self-replication of the most primitive HSC produces daughter cells that possess a long (possibly unlimited) clonal lifespan, while differentiation of HSCs results in a loss of such multilineage potential, and corresponding lineage commitment with a progressive reduction in their clonal lifespan. Previous studies indicate that the proliferation of HSC ex vivo appears to favor differentiation at the expense of self-replication, eventually resulting in a complete loss of HSC. Previous studies indicated that survival of HSC ex vivo in the absence of growth factors is limited, resulting in a complete loss of HSC after about 0.5-4 days in culture (Ploemacher et al., 1993; Li et al., 1994).
In contrast, transplantation studies have shown that a single HSC can repopulate the marrow of a lethally irradiated mouse, demonstrating that self-renewal of HSC occurs in vivo, as indicated by transplantation studies wherein a single HSC repopulated the marrow of an immunodeficient mouse (Smith, et al., 1991: Osawa et al., 1996). In addition, repopulation of secondary (and tertiary) recipients, has been demonstrated (Dick et al., 1985; Jordan et al., 1990; Keller et al., 1990). HSC have also been demonstrated to be capable of repopulating non-hematopoietic human tissues, including but not limited to liver (Petersen et al., 1999) and neuronal tissue (Bjornson et al., 1999).
Clinical trials are underway using treatment regimens that includes high-dose chemotherapy and/or radiation therapy together with bone marrow transplantation or transplantation of an HSC-containing cell population for the treatment of various cancers, including ovarian cancer, thymomas, germ cell tumors, multiple myeloma, melanoma, testicular cancer, lung cancer, and brain cancer.
Cell preparations enriched for hematopoietic stem cells generally contain a low percentage of cells capable of long-term hematopoietic reconstitution. In general, culture conditions effective to promote the survival of hematopoietic stem cells include cytokines, which stimulate cell division and differentiation of the cells, diminishing their long term repopulating capability. Frequently, as a result, in vivo administration of such cell preparations does not result in rapid repopulation of the host hematopoietic system. In particular, the slow repopulation of the neutrophil and platelet compartments of the hematopoietic system complicates the recovery process and may result in susceptibility to infection and/or complications due to poor blood clotting.
Transforming growth factor beta-1 (TGF-β1) is known to directly and reversibly inhibit the initial cell divisions of long-term repopulating hematopoietic stem cells (LTR-HSC) in vitro. (See, e.g., Sitnicka et al, 1996 and Ploemacher et al., 1993; Ottmann et al., 1988; Cashman et al., 1990.) The in vivo administration of TGF-β to humans to enhance the number of hematopoietic progenitor cells in peripheral blood has also been described. (See, e.g. U.S. Pat. No. 5,674,843, issued Oct. 7, 1997.) In addition, the release of CD34 positive human hematopoietic progenitor cells from quiescence has been reported following treatment with a phosphorothioate antisense oligomer to TGF-β1 or Rb1 in culture medium in the presence of cytokines and/or growth factors (Hatzfeld et al., 1991).
Although inhibition of genes associated with cellular development has been achieved using antisense technology, naturally occurring oligonucleotides have a nuclease-sensitive phosphodiester backbone. However, it has also been demonstrated that naturally occurring oligonucleotides can be modified rendering them resistant to degradation by nucleases, e.g., by incorporating a methylphosphonate, phosphorothioate or phosphodiamidate linkage into the oligonucleotide sequence in place of the standard phosphodiester linkage (Spitzer and Eckstein, 1988; Baker, et al, 1990; Hudziak, 1996).
For therapeutic purposes, it would be desirable to be able to regulate the differentiation of hematopoietic stem cells in vitro (ex vivo) and in vivo using an agent which acts specifically on hematopoietic stem cells and which is not sensitive to enzymatic degradation in cell culture or in vivo.